Role of cell membrane composition in receptor-mediated internalization of vesicular stomatitis virus in human HEp-2 cells

The role of essential fatty acids in membrane functions related to receptor-mediated endocytosis of vesicular stomatitis virus (VSV) was investigated using a human laryngeal carcinoma cell line (HEp-2) grown in chemically defined serum-free medium (DM) to deplete their essential fatty acid contents. VSV replicated much less effectively in HEp-2 cells grown in DM as compared to serum containing complete medium (CM). Observed reduction in the rate of virus multiplication was, at least in part, due to reduced virus penetration which was monitored using VSV labeled with nitroxyl free radicals as electron spin probe. Surface proteins of VSV were labeled with maleimide spin-label, and succinimide spin-label. Ni2+ was used as a broadening agent to identify the spin-label signals from viruses inside the cell. HEp-2 cells and mouse leukemia cell line L1210 treated with 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) cadaverine, an agent previously shown to inhibit the uptake of VSV in vitro, was used as a positive control in some experiments. VSV penetrated less effectively in both DM-grown cells and in CM-grown cells in the presence of dansylcadaverine. Similar results were obtained by monitoring the uptake of 125I-labeled VSV. When HEp-2 cells grown for several generations in DM were incubated with 10% fetal calf serum for 16 h, the cells supported virus replication to a similar extent as the cells grown in CM. In contrast, addition of arachidonic acid restored VSV growth only partially. Continued growth of HEp-2 cells in DM resulted in a shift in fatty acyl chain composition of phospholipids. The results indicate a finite role for essential fatty acids in receptor-mediated internalization of virus particles.     

Saturable binding sites for vesicular stomatitis virus on the surface of Vero cells.

The binding of vesicular stomatitis virus (VSV) to Vero monkey cells was studied by using virus metabolically labeled with [35S]methionine. Under conditions where viral uptake did not occur (4 degrees C), apparent binding equilibrium was achieved within 12 h at a level representing 12% of the input virus. Two distinct forms of virus-cell interaction were found. At low concentrations of VSV, corresponding to multiplicities used for tissue culture studies, saturable binding was the major form of interaction. Saturation was complete at approximately 4,000 VSV virions per cell. At higher virus concentrations, nonsaturable binding prevailed. Trypsin treatment of Vero cells did not decrease the binding of VSV to the saturable binding sites. Internalization of VSV at 37 degrees C also displayed a saturable component which was directly comparable to that observed for binding. VSV binding to high-affinity, saturable sites on the plasma membrane may represent a receptor-mediated route of viral uptake.     

Structural changes in BHK cell plasma membrane caused by the binding of vesicular stomatitis virus

Spin label electron spin resonance techniques using a nitroxide derivative of stearic acid were used to detect changes in plasma membrane structure caused by the binding of vesicular stomatitis virus (VSV) to cell plasma membranes of intact BHK-21 cells. The results indicate that binding of VSV to cell surface receptors causes an increase in the observed rigidity of the plasma membrane lipid bilayer. This change in membrane structure, which appears to be caused by the cross-linking of receptors in the plane of the plasma membrane, could be prevented by treating the cells with colchicine before addition of virus and could be reversed by treating the cells with colchicine after addition of virus. Cells treated with a monovalent, water-soluble derivative of VSV G-protein (Gs) did not show an increase in plasma membrane bilayer rigidity. However, addition of anti-VSV G-protein immunoglobulin G to cells pretreated with G8 caused an increase in plasma membrane bilayer rigidity. This increased rigidity could also be reversed by the addition of colchicine. Fluorescence microscopy was used to determine the distribution of fluorescein-labeled VSV particles on the cell surface after addition of virus. Approximately 30 min after addition of virus, discrete areas on the cell surface showed fluorescent staining, which coalesced to apical regions of the cell after approximately 40 min.     

Effect of membrane protein on lipid bilayer structure: a spin-label electron spin resonance study of vesicular stomatitis virus.

Spin-label electron spin resonance (ESR) methods have been used to study the structure of the envelope of vesicular stomatitis virus (VSV). The data indicate that the lipid is organized in a bilayer structure. Proteolytic digestion of the glycoproteins which are the spike-like projections on the outer surface of the virus particle increases the fluidity of the lipid bilayer. Since the lipid composition of the virion reflects the composition of the host plasma membrane and the protein composition is determined by the viral genome, VSV was grown in both MDBK and BHK21-F cells to determine the effect of a change in lipid composition on the structure of the lipid bilayer of VSV. The lipid bilayer of the virion was found to be more rigid when derived from MDBK cells than from BHK21-F cells. Studies comparing spin-labeled intact cells and cell membrane fractions suggest that upon labeling the whole cell the spin label probes the plasma membrane. Comparison of spin-labeled VSV particles and their host cells indicates that the lipid bilayer of the plasma membrane is considerably more fluid than that of the virion. These results are discussed in terms of the effect of membrane-associated protein on the structure of the lipid bilayer.     

Analysis of the role of p200-containing vesicles in post-Golgi traffic.

p200 is a cytoplasmic protein that associates with vesicles budding from the trans-golgi network (TGN). The protein was identified by a monoclonal antibody AD7. We have used this antibody to analyze whether p200 functions in exocytic transport from the TGN to the apical or basolateral plasma membrane in Madin-Darby canine kidney cells. We found that transport of the viral marker proteins, influenza hemagglutinin (HA) to the apical surface or vesicular stomatitis virus glycoprotein (VSV G) to the basolateral surface in streptolysin O-permeabilized cells was not affected when p200 was depleted from both the membranes and the cytosol. When vesicles isolated from perforated cells were analyzed by equilibrium density gradient centrifugation, the p200 immunoreactive membranes did not comigrate with either the apical vesicle marker HA or the basolateral vesicle marker VSV G. Immunoelectron microscopy of perforated and double-labeled cells showed that the p200 positive vesicular profiles were not labeled by antibodies to HA or VSV G when the viral proteins were accumulated in the TGN. Furthermore, the p200-decorated vesicles were more electron dense than those labeled with the viral antibodies. Together, these results suggest that p200 does not function in the transport pathways that carry HA from the TGN to the apical surface or VSV G from the TGN to the basolateral surface.     

Effect of erythrocyte transbilayer phospholipid distribution on fusion with vesicular stomatitis virus

To identify the specific component(s) in the target membrane involved in fusion of vesicular stomatitis virus (VSV), we examined the interaction of the virus with human erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion was monitored spectrofluorometrically by the octadecylrhodamine dequenching assay. Fusion of VSV with lipid-symmetric erythrocyte ghosts was rapid at 37 degrees C and low pH, whereas little or no fusion was observed with lipid-asymmetric ghosts. Conversion of phosphatidylserine in the lipid-symmetric ghost membrane to phosphatidylethanolamine by means of the enzyme phosphatidylserine decarboxylase did not alter the target membrane's susceptibility to VSV fusion. Spin-labeled phospholipid analogues with phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine headgroups incorporated into the outer leaflet of lipid-asymmetric erythrocytes did not render those membranes fusogenic. Electron spin resonance spectra showed an increased mobility of a phosphatidylcholine spin-label incorporated into the outer leaflet of lipid-symmetric erythrocyte ghosts as compared to that of lipid-asymmetric ghosts. These results indicate that the susceptibility to VSV fusion is not dependent on any particular phospholipid but rather is related to packing characteristics of the target membrane.     

Introduction of cationic virosome derived from vesicular stomatitis virus as a novel gene delivery system for sf9 cells

Insect-derived cell lines are used extensively to produce recombinant proteins because they are capable of performing a range of post-translational modifications. Due to their significance in biotechnological applications, various methods have been developed to transfect them. In this study, we introduce a virosome constructed from vesicular stomatitis virus (VSV) as a new delivery system for sf9 cells. We labeled these VSV virosomes by fluorescent probe Rhodamine B chloride (R18). By fluorescence microscope observation and conducting a fusion assay, we confirmed the uptake of VSV virosomes via endocytosis by sf9 cells and their fusion with the endosomal membrane. Moreover, we incubated cationic VSV virosomes with a GFP-expressing bacmid and transfected sf9 cells, after 24?h some cells expressed GFP indicating the ability of VSV virosomes to deliver heterologous DNA to these cells. This is the first report of a virosome-based delivery system introduced for an insect cell line.     

Pyrene phospholipid as a biological fluorescent probe for studying fusion of virus membrane with liposomes

We are using fluorescent endogenous phospholipids in virus membranes to study the factors that promote fusion on interaction with receptor membranes. To this end, vesicular stomatitis virus (VSV) grown in baby hamster kidney (BHK-21) cells was biologically labeled with fluorescent lipids, primarily phosphatidylcholine and phosphatidylethanolamine, derived from pyrene fatty acids. The pyrene lipids present in the virions showed a fluorescence spectrum typical of pyrene with an intense monomer and a broad excimer. Interaction of pyrene lipid labeled VSV with serum lipoproteins led to a spontaneous fast transfer of the small amount of pyrene fatty acids present in the envelope (t1/2 less than or equal to 7 min), followed by a considerably slower transfer of pyrene phospholipids from the membrane of the virions (t1/2 greater than or equal to 12 h). Incubation of pyrene phospholipid labeled VSV with phosphatidylserine small unilamellar vesicles resulted in fusion at low pH (pH 5.0) as measured by the change in the excimer/monomer fluorescence intensity ratio. Fusion kinetics was rapid, reaching a plateau after 4 min at pH 5.0 and 37 degrees C. Only negligible fusion was noted at neutral pH or at 4 degrees C. Fully infectious virions labeled biologically with fluorescent lipids provide a useful tool for studying mechanisms of cell-virus interactions and neutralization of viral infectivity by specific monoclonal antibodies reactive with viral membrane glycoprotein.     

Purification and some properties of human erythrocyte calpastatin

By means of a new type of microinjection apparatus, which has a micropipette located in a hole through the optical axis of the condenser lens, we injected interferon (IFN) or 2',5'-oligoadenylate (2-5A) into mouse L cells, and observed their antiviral effects on the multiplication of vesicular stomatitis virus (VSV). After injection, cells were infected with VSV, and labeled with [3H]uridine in the presence of actinomycin D. The proportion of cells infected with VSV which carried radioactive virus-RNA was determined by autoradiography. IFN introduced directly into L cells had no effect on the virus growth. This result supports the idea that IFN molecules exert their effect from outside the cell membrane without penetrating into the cytoplasm. 2-5A, on the other hand, was able to inhibit the growth of VSV effectively when injected into L cells. The antiviral effect was dependent on the dose of 2-5A injected, and moreover the effect was transient, since it disappeared completely after 24-h incubation.     

Role of viral envelope sialic acid in membrane fusion mediated by the vesicular stomatitis virus envelope glycoprotein.

Fusion of vesicular stomatitis virus (VSV) with cells and liposomes before and after treatment with neuraminidase was studied using the R18 dequenching assay. Desialylation of VSV significantly enhanced the extent of fusion with Vero cells but affected neither the pH dependence nor the binding of VSV to Vero cells. The enhanced fusion of asialo-VSV was observed both at the plasma membrane as well as via the endocytic pathway. Both VSV and asialo-VSV fused with liposomes made of neutral phospholipid, but only asialo-VSV fused with liposomes containing a 1:1 mixture of neutral and negatively charged phospholipid. To examine factors which contribute to the extent of fusion, we analyzed the various activation and inactivation reactions that take place as a result of low-pH triggering of VSV prebound to the target membrane. Lag times for the onset of fusion were similar for VSV and asialo-VSV, indicating that desialylation did not affect the activation reactions. However, exposure of VSV bound to target membranes at pH 6.5 for 400 s led to considerable inactivation, whereas little inactivation was seen after desialylation of VSV. These results are analyzed in terms of a model which allows us to determine which components of the overall fusion process are dominated by viral envelope sialic acid.     

Viral glycoproteins destined for apical or basolateral plasma membrane domains traverse the same Golgi apparatus during their intracellular transport in doubly infected Madin-Darby canine kidney cells

Madin-Darby canine kidney (MDCK) cells can sustain double infection with pairs of viruses of opposite budding polarity (simian virus 5 [SV5] and vesicular stomatitis virus [VSV] or influenza and VSV), and we observed that in such cells the envelope glycoproteins of the two viruses are synthesized simultaneously and assembled into virions at their characteristic sites. Influenza and SV5 budded exclusively from the apical plasma membrane of the cells, while VSV emerged only from the basolateral surfaces. Immunoelectron microscopic examination of doubly infected MDCK cells showed that the influenza hemagglutinin (HA) and the VSV G glycoproteins traverse the same Golgi apparatus and even the same Golgi cisternae. This indicates that the pathways of the two proteins towards the plasma membrane do not diverge before passage through the Golgi apparatus and therefore that critical sorting steps must take place during or after passage of the glycoproteins through this organelle. After its passage through the Golgi, the HA accumulated primarily at the apical membrane, where influenza virion assembly occurred. A small fraction of HA did, however, appear on the lateral surface and was incorporated into the envelope of budding VSV virions. Although predominantly found on the basolateral surface, significant amounts of G protein were observed on the apical plasma membrane well before disruption of the tight junctions was detectable. Nevertheless, assembly of VSV virions was restricted to the basolateral domain and in doubly infected cells the G protein was only infrequently incorporated into the envelope of budding influenza virions. These observations indicate that the site of VSV budding is not determined exclusively by the presence of G polypeptides. Therefore, it is likely that, at least for VSV, other cellular or viral components are responsible for the selection of the appropriate budding domain.     

Correlations between fatty acid distribution in phospholipids and the temperature dependence of membrane physical state

Cytoplasmic membranes of an unsaturated fatty acid auxotroph of Escherichia coli have been studied using spin labeled hydrocarbon probes. These studies reveal that the membrane lipids undergo changes of state at critical temperatures which reflect the physical properties of the fatty acid supplement supplied to the cells during growth. The critical temperatures observed in spin labeled membranes correlate with characteristic temperatures in membrane functions. Lipid analysis reveals that fatty acid composition and distribution in membrane phospholipids are primary determinants of the temperatures at which changes of state are observed in membrane lipids. Fatty acid composition and distribution can also produce unique interactions between certain spin label probes and their lipid environment.     

Viruses budding from either the apical or the basolateral plasma membrane domain of MDCK cells have unique phospholipid compositions.

Influenza virus and vesicular stomatitis virus (VSV) obtain their lipid envelope by budding through the plasma membrane of infected cells. When monolayers of Madin-Darby canine kidney (MDCK) cells, a polarized epithelial cell line, are infected with fowl plague virus (FPV), an avian influenza virus, or with VSV, new FPV buds through the apical plasma membrane whereas VSV progeny is formed by budding through the basolateral plasma membrane. FPV and VSV were isolated from MDCK host cells prelabeled with [32P]orthophosphate and their phospholipid compositions were compared. Infection was carried out at 31 degrees C to delay cytopathic effects of the virus infection, which lead to depolarization of the cell surface. 32P-labeled FPV was isolated from the culture medium, whereas 32P-labeled VSV was released from below the cell monolayer by scraping the cells from the culture dish 8 h after infection. At this time little VSV was found in the culture medium, indicating that the cells were still polarized. The phospholipid composition of the two viruses was distinctly different. FPV was enriched in phosphatidylethanolamine and phosphatidylserine and VSV in phosphatidylcholine, sphingomyelin, and phosphatidylinositol. When MDCK cells were trypsinized after infection and replated, non-infected control cells attached to reform a confluent monolayer within 4 h, whereas infected cells remained in suspension. FPV and VSV could be isolated from the cells in suspension and under these conditions the phospholipid composition of the two viruses was very similar. We conclude that the two viruses obtain their lipids from the plasma membrane in the same way and that the different phospholipid compositions of the viruses from polarized cells reflect differences in the phospholipid composition of the two plasma membrane domains.     

Isolation and characterization of tunicamycin resistant mutants from Chinese hamster ovary cells

Stable clones selected for resistance to tunicamycin (TM) have been isolated from Chinese Hamster Ovary (CHO) cells. The TMR phenotype is stable for more than nine months in the absence of the drug. The morphology of TMR mutant varies from epitheloid to abnormally elongate. The mutants do not display cross-resistance for ConA but are slightly cross-resistant to PHA. Biochemically labeled membrane proteins and glycoprotein of Vesicular stomatitis virus (VSV) grown in the TMR mutants revealed that the incorporation of radioactive glucosamine was markedly reduced in the mutants. The results indicate that TMR cells are a novel type of membrane mutant.     

Specific incorporation of host cell surface proteins into budding vesicular stomatitis virus particles

The specific incorporation of cell surface proteins into budding Vesicular Stomatitis Virus (VSV) particles was shown by two approaches. In the first, monolayer cultures of Vero or L cells were labeled by lactoperoxidase-catalyzed iodination and the cells were then infected with VSV. Approximately 2% of the cell surface ¹²⁵1 radioactivity was incorporated into particles which co-purify with normal, infectious virions by both velocity and equilibrium gradient centrifugation and which are precipitated by antiserum specific for the VSV glycoprotein. Control experiments establish that these ¹²⁵I-labeled particles are not cell debris or cellular material which aggregate with or adhere to VSV virions. VSV virions contain only a subset of the 10–15 normal ¹²⁵1-labeled cell surface polypeptides resolved by SDS gel electrophoresis; VSV grown in L cells and Vero cells incorporate different host polypeptides. In a second approach, Vero cells were labeled with ³⁵S-methione, then infected with VSV. Two predominant host polypeptides (molecular weights 110,000 and 20,000) were incorporated into VSV virions. These proteins, like VSV G protein, are exposed to the surface of the virion. They co-migrate with the major incorporated ¹²⁵1 host polypeptides. These host proteins are present in approximately 10 and 80 copies, respectively, per virion. Specific incorporation of host polypeptides into VSV virions does not require the presence of viral glycoprotein. This was shown by use of a ts VSV mutant defective in maturation of VSV G protein to the cell surface. Budding from infected cells are noninfectious particles which contain all the viral proteins except for G; these particles contain the same proportion and spectrum of ¹²⁵1-labeled host surface polypeptides as do wild-type virions. These results extend previous conclusions implicating the submembrane viral matrix protein, or the viral nucleocapsid, as being of primary importance in selecting cell surface proteins for incorporation into budding VSV virions.     

pH-dependent fusion of reconstituted vesicular stomatitis virus envelopes with Vero cells. Measurement by dequenching of fluorescence

Reconstituted vesicular stomatitis virus (VSV) envelopes were formed by solubilization of the viral envelope with Triton X-100 followed by removal of detergent by direct addition of SM2 biobeads. We provide direct demonstration of fusion of reconstituted VSV with cells using fluorescent lipid and aqueous probes incorporated into the VSV virosomes during reconstitution. We show a direct comparison of the kinetics and pH profile of fusion with cells between reconstituted VSV and fluorescently labeled intact virus. With this preparation it is now possible to gain additional information about the role of cooperativity in viral protein-mediated fusion, and to permit construction of efficient vehicles for delivery of drugs and other materials into cells.     

The M protein of vesicular stomatitis virus associates specifically with the basolateral membranes of polarized epithelial cells independently of the G protein

Using monoclonal antibodies and indirect immunofluorescence microscopy, we investigated the distribution of the M protein in situ in vesicular stomatitis virus-(VSV) infected MDCK cells. M protein was observed free in the cytoplasm and associated with the plasma membrane. Using the ts045 mutant of VSV to uncouple the synthesis and transport of the VSV G protein we demonstrated that this distribution was not related to the presence of G protein on the cell surface. Sections of epon-embedded infected cells labeled with antibody to the M protein and processed for indirect horseradish peroxidase immunocytochemistry revealed that the M protein was associated specifically with the basolateral plasma membrane. The G and M proteins of VSV have therefore evolved features which bring them independently to the basolateral membrane of polarized epithelial cells and allow virus to bud specifically from that membrane.     

Depolymerization of microtubules alters membrane potential and affects the motional freedom of membrane proteins

Two independent lines of evidence were obtained indicating that microtubule depolymerization affects the functions and the physical state of membranes in intact Chinese hamster ovary cells. The first type of evidence was obtained by using the dye dihexyloxacarbocyanine iodide to measure membrane potential before and after treatment with several microtubule active agents. Microtubule depolymerization resulted in a decrease in cell fluorescence, whereas stabilization of microtubules with taxol resulted in an increase in cell fluorescence. These effects of the drugs were due to their interactions with microtubules and not to direct effects of the drugs on the plasma membranes for the following reasons: effects were time dependent and required entry into the cells as indicated by the lack of fluorescence change in a multi-drug-resistant mutant that does not accumulate antimicrotubule drugs and a colcemid-resistant tubulin mutant did not show these effects on cell fluorescence. Evidence for altered motional freedom of membrane proteins in the plasma membrane was obtained by using electron spin resonance analysis of maleimide spin probe labeled cells. This study showed that depolymerization of microtubules results in increased motional freedom of maleimide-labeled sulfhydryl group containing proteins. Taken together, these data argue that microtubules function in mammalian cells to regulate the physical state of membranes and modulate membrane potential generated across cell membranes.